Gene targeting—the process of gene replacement by homologous recombination or mutation—is a very useful, but typically inefficient technique for introducing desired changes in the genetic material of a host cell. Only when powerful selection for the targeted product can be applied is recovery of the desired alteration possible. A general method for improving the efficiency of gene targeting would be valuable in many circumstances, as would extension of this tool to a broader range of organisms.
It has been demonstrated in model experiments that introduction of a double-strand break (DSB) in host DNA greatly enhances the frequency of localized recombination. However, those tests required insertion of a recognition site for a specific endonuclease before cleavage could be induced. Similarly, in Drosophila the DSBs produced by P-element excision are recombinagenic, but require the P-element to preexist at the target site.
Although previously demonstrated methods of genetic transformation had been highly successful, transformation without targeted recombination has also been accompanied by problems associated with random insertion of the introduced DNA. Random integration can lead to the inactivation of essential genes, or to the aberrant expression of the introduced gene. Additional problems associated with genetic transformation include mosaicism due to multiple integrations, and technical difficulties associated with generation of replication defective recombinant viral vectors.
Targeted genetic recombination or mutation of a cell or organism is now possible because complete genomic sequences have been determined for a number of organisms, and more sequences are being obtained each day. Not only would the ability to direct a mutation to a specific genetic locus greatly aid those studying the function of particular genes, targeted genetic recombination would also have therapeutic and agricultural applications. Methods of targeted genetic recombination are needed that are more general, efficient, and/or reproducible than currently available techniques.